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. 2017 Jun 19;12(6):e0179860.
doi: 10.1371/journal.pone.0179860. eCollection 2017.

MicroRNA-433-3p promotes osteoblast differentiation through targeting DKK1 expression

Xiaolin Tang  1 , Jiantao Lin  2 , Guanhai Wang  2 , Jianlin Lu  1
Affiliations

MicroRNA-433-3p promotes osteoblast differentiation through targeting DKK1 expression

Xiaolin Tang et al. PLoS One. .

Abstract

Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/β-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Correlation of serum DKK1 with circulating miR-433-3p.
Up-regulated DKK1 level was found in osteoporotic rats compared to sham control rats (A, n = 6, U-test, p<0.01). Correlation of serum DKK1 with circulating miR-433-3p (B, linear regression, r = 0.7520, p = 0.046).
Fig 2
Fig 2. Validation of miR-433-3p targeting DKK1 mRNA 3’UTR.
Schematic figure showed miR-433-3p targeting DKK1 mRNA 3'UTR, mapping from site 554 to 560 (A). In the luciferase reporter assay, data showed vector constructed DKK1 mRNA 3'UTR group had quite lower luminescence signal in the present of miR-433-3p siRNA co-transfection simultaneously, whereas binding site mutant group had no reduction in luminescence signal (B, ANOVA, p<0.001, Mean ± SE of triple experiments with four duplicate wells). Protein immunoblotting assay showed miR-433-3p inhibited DKK1 expression up to 90% of total protein (C).
Fig 3
Fig 3. Effects of miR-433-3p on osteoblasts differentiation and maturation.
Lentivirus mediated miR-433-3p stable expression increased ALP activity of immortalized human hFOB1.19 and rat ROS 17/2.8 cells (A, p<0.01, triple experiments). miR-433-3p also increased primary bone marrow induced ostetoblasts ALP activity, but not in miR-433-3p plus sponge vector group (B, **, p<0.01). Infection of lentivirus miR-433-3p to human hFOB1.19 cells showed increased mRNA level of osteogenic markers RUNX2 and osteocalcin (C, **, p<0.01). Alizarin red staining results indicated that miR-433-3p promoted Ca2+ deposited within osteoblasts (D, p<0.01). Bar chart showed ten counting of views under microscopy of each group.
Fig 4
Fig 4. miRNA-433-3p quantification and staining in bone tissue.
Data shown primary cultured osteoblasts had higher miR-433-3p level compared with immortal cells using real-time PCR(A, p< 0.01). Red cells showed osteoblasts by ALP staining in decalcified bone tissues (B). DIG-labeled antisense of miR-433-3p probe was detected by anti-DIG secondary antibody, thus red color was observed under microscopy with 510–530 nm excitation (C, Rhodamine labeled, p<0.05).
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References

    1. Zhang W, Drake MT. Potential role for therapies targeting DKK1, LRP5, and serotonin in the treatment of osteoporosis. Curr Osteoporos Rep, 2012; 10(1): 93–100. doi: 10.1007/s11914-011-0086-8 - DOI - PubMed
    1. David R, Brenner C, Stieber J, Schwarz F, Brunner S, Vollmer M, et al. MesP1 drives vertebrate cardiovascular differentiation through Dkk-1-mediated blockade of Wnt-signalling. NAT CELL BIOL, 2008; 10(3): 338–45. doi: 10.1038/ncb1696 - DOI - PubMed
    1. Li J, Sarosi I, Cattley RC, Pretorius J, Asuncion F, Grisanti M, et al. Dkk1-mediated inhibition of Wnt signaling in bone results in osteopenia. BONE, 2006; 39(4): 754–66. doi: 10.1016/j.bone.2006.03.017 - DOI - PubMed
    1. Morvan F, Boulukos K, Clement-Lacroix P, Roman RS, Suc-Royer I, Vayssiere B, et al. Deletion of a single allele of the Dkk1 gene leads to an increase in bone formation and bone mass. J BONE MINER RES, 2006; 21(6): 934–45. doi: 10.1359/jbmr.060311 - DOI - PubMed
    1. Florio M, Gunasekaran K, Stolina M, Li X, Liu L, Tipton B, et al. A bispecific antibody targeting sclerostin and DKK-1 promotes bone mass accrual and fracture repair. NAT COMMUN, 2016; 7: 11505 doi: 10.1038/ncomms11505 - DOI - PMC - PubMed

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